Regulatory T cells (Tregs) are critical for proper control of the immune response and play a clear role in regulation of autoimmune disease. Further highlighting their importance, Tregs provide an antigen specific therapy that is currently being evaluated in preclinical and clinical trials. Although they are of unquestioned value, it is difficult to assess Treg specificity for antigen. In fact, many experimental protocols make use of non-specific TCR stimuli such as anti-CD3/anti-CD28 for their activation, and typical assessments of specificity such as peptide: MHC Class II tetramers and functional readouts are not especially effective when applied to Tregs. As TCR specificity and affinity are crucial to any T cell functional response, we need to understand what affinity range works best for optimal Treg use as well as for their differentiation. Our lab is in a unique position to examine questions on TCR specificity and affinity because we have developed a novel assay system based on two dimensional (2D) micropipette technology that provides the most sensitive means currently available to assess these parameters. The prevalent view of thymic derived Tregs (tTreg) in terms of affinity is that their repertoires are comprised of the highest affinity TCRs that escaped negative selection; however, our preliminary data has identified a range of affinities of tTregs specific for myelin oligodendrocyte glycoprotein (MOG). Instead of driving thymocyte negative selection, MOG supports development of thymic derived MOG specific Tregs. We propose that these Tregs will be of therapeutic use and based on our preliminary studies, propose the following two aims to track Treg specificity, location, kinetics, stability, and potency during chronic and relapsing/remitting demyelinating disease. Aim 1: To define MOG-specific Treg affinity and frequency during demyelinating disease- Aim 2: To determine Treg specificity and lineage stability for MOG-